An Overview of United States Pharmacopeia Sterility Testing
Sterility testing of compounded sterile preparations (CSPs) is only one part of the quality-control testing required to ensure that preparations have been prepared using strict aseptic processes. Since sterility testing only determines the actual sterility of the sample(s) tested, it is, therefore, essential to use adequate sample sizes and sample volumes to allow for the results to be representative of the entire batch. To help support the sterility-test results, the following practices should also be in place:
- Routine cleaning and disinfection of the aseptic work area to maintain International Organization for Standardization (ISO) Class-5 air quality
- Regular air-quality and surface testing of the direct compounding environment to ensure minimal microbial surface contamination
- Glove imprint testing to control the potential for contamination of the critical site
- Visual confirmation that compounding personnel are properly donning and wearing the appropriate items and types of protective garments
- Certification of technicians as to their knowledge and dexterity to perform scheduled tasks
- Certification of equipment (e.g., laminar airflow hoods, cleanrooms) using the appropriate standards
Requirements for Valid Sterility Testing
United States Pharmacopeia (USP) Chapter 71 for Sterility Tests outlines the various requirements for conducting testing. All manipulation of the samples and actual sterility testing should be done under aseptic conditions. This will help to avoid false positive results and further headaches down the line.
There are two types of broth that should be used for sterility testing:
- Soybean casein digest medium - detects the presence of aerobic bacteria and fungi when incubated at 22.5°C ± 2.5°C.
- Fluid thioglycollate medium - cultures anaerobic organisms when incubated at 32.5°C ± 2.5°C.
Should the test samples contain a penicillin or cephalosporin, steps must be taken to inactivate the antibiotic by addition of the appropriate β-lactamases to the medium, if using the direct inoculation method, or the rinse solution if the membrane filtration method is used. Solutions containing a preservative should be either neutralized or diluted out before testing.
To completely validate the test conditions above, a method-suitability test must be performed to eliminate the possibility that the CSP being tested has an inherent cidal or static property that will generate false-negative results. This test basically involves challenging the conditions by adding or filtering no more than 100 cfu of the organisms outlined in USP Chapter 71 after the CSP has been processed. All method-suitability test results must turn positive after 14 days of incubation to confirm that the CSP has no antimicrobial activity. If any of the organisms fail to thrive, then the method is not valid and the conditions have to be modified to eliminate the interference. The most common methods for overcoming the effects of growth inhibition are 1) using a larger volume of media to dilute the CSP or 2) inactivation as in the case of an antibiotic. Once all the results are positive, the method is validated, and the new conditions have to be incorporated into all future sterility testing. If there is a change to the composition of the CSP, then the sterility test has to be revalidated.
All media used should have a growth promotion challenge conducted for aerobic, anaerobic, and fungi using the organism listed in USP Chapter 71 to confirm the media is able to sustain growth of the organisms. This should be performed in-house for every batch of media prepared, or conducted by the manufacturer of the media and reported on the release certificate. As well, a negative control should be incorporated into the testing to ensure the media was sterile to begin with and to help eliminate possible false positive results.
There are two official USP sterility-test methods that can be used for testing of CSPs. The first consists of filtering the solution through a membrane of not greater than 0.45 micron to collect any organisms that may be present. The filters are then rinsed, no less than three times, with the volume of sterile diluent determined in the method-suitability test. After rinsing, a whole or half of a membrane should be placed into suitable containers of sterile media. For small volumes, the contents of multiple containers may be pooled in a sterile vessel before filtering. The USP gives instructions for sterility testing of soluble solids, oils and oily solutions, ointments and creams, and prefilled syringes when using the membrane-filtration technique.
The second method involves direct inoculation of the sample into sterile culture media. For this method, the volume of sample should not be more than 10% of the volume of media used. The procedure for dealing with oily solutions, ointments and creams, solids, and sterile devices are outlined in Chapter 71 of the USP.
All containers of test media should be incubated at the designated temperature for a total of 14 days. During the incubation period, the solutions should be monitored periodically for the development of turbidity, indicating bacterial growth. If the test sample affects the clarity of the media, when using the direct inoculation method, then after 14 days incubation, a 1-mL aliquot may be removed, placed into fresh media, and incubated for no less than 4 days. The appearance of no microbial growth then confirms the sterility of the samples.
The minimum volume of sample per container for each medium and the minimum number of articles to be tested per batch size are provided in Chapter 71 of the USP. CSPs are categorized into three groups based primarily on the potential for microbial contamination during the compounding activity. These levels are: 1) low-risk, 2) medium-risk, and 3) high-risk. In the absence of performing a sterility test, low-risk CSPs, compounded aseptically, can be assigned a beyond-use date (BUD) of no more than 48 hours at controlled room temperature, or 14 days at a 2°C to 8°C, or 45 days when in the frozen state between -10°C and -25°C. Medium-risk CSPs may be kept for not more than 30 hours at controlled room temperature, or 9 days at a 2°C to 8°C, or 45 days when stored between -10°C and -25°C without performing a sterility test. For a sterilized high-risk level preparation, the BUD cannot exceed 24 hours when stored in a controlled room temperature environment, or 3 days at 2°C to 8°C, or 45 days when frozen solid between -10°C and -25°C if a sterility test has not been completed. In addition, media-fill challenge testing, which assesses the skill of personnel to aseptically prepare CSPs, should be conducted at least annually for low- and medium-risk level products and semi-annually for high-risk level activities.
Interpretation of Results
If the media remains clear, indicating no bacterial growth, then the CSP complies with the test for sterility. However, if the media indicates the presence of bacterial growth, by turning turbid, at any point during the 14-day incubation period, then the CSP fails the test for sterility, unless the test is deemed invalid for causes unrelated to the preparation.
To have the test results deemed invalid, one of the following must have occurred:
- The microbial monitoring of the test facility failed.
- A fault in the testing procedure was discovered during an investigation.
- The negative control showed signs of bacterial growth.
- Identification of the organisms in the failed test media can unequivocally be attributed to faults with materials or the testing methods used.
If the test is declared invalid, the testing can be repeated using the same number of articles as the original testing. If the repeat testing shows no sign of bacterial growth after 14 days, then the CSP complies with the test for sterility. If bacteria are present in the repeat test, then the CSP fails the test for sterility.
Without performing sterility testing using a validated method, the BUDs of CSPs are limited to the guidelines given in the USP. Once the preparations pass the sterility testing, BUDs can be extended based on other factors such as the stability of the individual ingredients and expiry dates of the packaging supplies used in preparing the preparations.
Ronald F. Donnelly, MSc, BSc
The Ottawa Hospital (Civic Campus)